Method and compositions for the synthesis of dioxolane nucleosides with β configuration

ABSTRACT

The present invention relates to methods and compositions for preparing biologically important nucleoside analogues containing 1,3-dioxolane sugar rings. In particular, this invention relates to the stereoselective synthesis of the beta (cis) isomer by glycosylating the base with an intermediate of formula (II) below a temperature of about -10° C. ##STR1## wherein R 1  and L are defined herein.

This application is a 371 of PCT/CA96/00845 filed Dec. 13, 1996.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for preparingnucleoside analogues containing dioxolane sugar rings. In particular,the invention relates to the stereoselective synthesis 1,3-dioxolanenucleosides having β or cis configuration.

BACKGROUND OF THE INVENTION

Nucleosides and their analogues represent an important class ofchemotherapeutic agents with antiviral, anticancer, immunomodulatory andantibiotic activities. Nucleoside analogues such as3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxyinosine (ddI),2',3'-dideoxycytidine (ddC), 3'-deoxy-2',3'-didehydrothymidine (d₄ T)and (-)-2'-deoxy-3'-thiacytidine (3TC™) are clinically approved for thetreatment of infections caused by the human immunodeficiency viruses.2'-Deoxy-2'-methylidenecytidine (DMDC, Yamagami et al. Cancer Research1991, 51, 2319) and 2'-deoxy-2',2'-difluorocytidine (gemcytidine, Hertelet al. J. Org. Chem. 1988, 53, 2406) are nucleoside analogues withantitumor activity. A number of C-8 substituted guanosines such as7-thia-8-oxoguanosine (Smee et al. J. Biol. Response Mod. 1990, 9, 24)8-bromoguanosine and 8-mercaptoguanosine (Wicker et al. Cell Immunol.1987, 106, 318) stimulate the immune system and induce the production ofinterferon. All of the above biologically active nucleosides are singleenantiomers.

Recently, several members of the 3'-heterosubstituted class of2',3'-dideoxynucleoside analogues such as 3TC™(Coates et al. Antimicrob.Agents Chemother. 1992, 36, 202), (-)-FTC (Chang et al. J. Bio. Chem.1992, 267, 13938-13942) (-)-dioxolane C (Kim et al. Tetrahedron Lett.1992, 33, 6899) have been reported to possess potent activity againstHIV and HBV replication and possess the β-L absolute configuration.(-)-Dioxolane C has been reported to possess antitumor activity (Groveet al. Cancer Res. 1995, 55, 3008-3011). The dideoxynucleoside analogues(-)-dOTC and (-)-dOTFC (Mansour et al. J. Med. Chem. 1995, 38, 1-4) wereselective in activity against HIV-1.

For a stereoselective synthesis of nucleoside analogues, it is essentialthat the nucleobase be introduced predominately with the desiredrelative stereochemistry without causing anomerization in thecarbohydrate portion. One approach to achieve this is to modify thecarbohydrate portion of a preassembled nucleoside by a variety ofdeoxygenation reactions (Chu et al. J. Org. Chem. 1989, 54, 2217-2225;Marcuccio et al. Nucleosides Nucleotides 1992, 11, 1695-1701; Starrettet al. Nucleosides Nucleotides 1990, 9, 885-897, Bhat et al. NucleosidesNucleotides 1990, 9, 1061-1065). This approach however is limited to thesynthesis of those analogues whose absolute configuration resembles thatof the starting nucleoside and would not be practical if lengthyprocedures are required to prepare the starting nucleoside prior todeoxygenation as would be the case for β-L dideoxynucleosides. Analternative approach to achieve stereoselectivity has been reportedwhich requires assembling the nucleoside analogue by a reaction of abase or its synthetic precursor with the carbohydrate portion underLewis acid coupling procedures or SN-2 like conditions.

It is well known in the art that glycosylation of bases to dideoxysugarsproceed in low stereoselectivity in the absence of a 2'-substituent onthe carbohydrate rings capable of neighboring group participation. Okabeet al. (J. Org. Chem. 1988, 53, 4780-4786) reported the highest ratio ofβ:α isomers of ddC of 60:40 with ethylaluminium dichloride as the Lewisacid. However, with a phenylselenenyl substituent at the C-2 position ofthe carbohydrate (Chu et al. J. Org. Chem. 1980, 55, 1418-1420; Beach etal. J. Org. Chem. 1992, 57, 3887-3894) or a phenylsulfenyl moiety(Wilson et al. Tetrahedron Lett. 1990, 31, 1815-1818) the β:α ratioincreases to 99:1. To overcome problems of introducing such substituentswith the desired α-stereochemistry, Kawakami et al. (NucleosidesNucleotides 1992, 11, 1673-1682) reported that disubstitution at C-2 ofthe sugar ring as in 2,2-diphenylthio-2,3-dideoxyribose affordsnucleosides in the ratio of β:α=80:20 when reacted with silylated basesin the presence of trimethylsilyltriflate (TMSOTf) as a catalyst.Although this strategy enabled the synthesis of the β-anomer, removal ofthe phenylthio group proved to be problematic.

Due to the limited generality in introducing the C-2 substituentstereoselectively, synthetic methodologies based on electrophilicaddition of phenyl sulfenyl halides or N-iodosuccinimides andnucleobases to furanoid glycal intermediates have been reported (Kim etal. Tetrahedron Lett. 1992, 33, 5733-5376; Kawakami et al. Heterocycles1993, 36, 665-669; Wang et al. Tetrahedron Lett. 1993, 34, 4881-4884;El-laghdach et al. Tetrahedron Lett. 1993, 34, 2821-2822). In thisapproach, the 2'-substituent is introduced in situ however, multistepprocedures are needed for removal of such substituents.

SN-2 like coupling procedures of 1-chloro and 1-bromo 2,3-dideoxysugarshave been investigated (Farina et al. Tetrahedron Lett. 1988, 29,1239-1242; Kawakami et al. Heterocycles 1990, 31, 2041-2053). However,the highest ratio of β to α nucleosides reported is 70:30 respectively.

In situ complexation of metal salts such as SnCl₄ or Ti(O-Pr)₂ Cl₂ tothe α-face of the sugar precursor when the sugar portion is anoxathiolanyl or dioxolanyl derivative produces β-pyrimidine nucleosides(Choi et al. J. Am. Chem. Soc. 1991, 113, 9377-9379). Despite the highratio of β- to α-anomers obtained in this approach, a serious limitationwith enantiomerically pure sugar precursor is reported leading toracemic nucleosides (Beach et al. J. Org. Chem. 1992, 57, 2217-2219;Humber et al. Tetrahedron Lett. 1992, 32, 4625-4628; Hoong et al. J.Org. Chem. 1992, 57, 5563-5565). In order to produce one enantiomericform of racemic nucleosides, enzymatic and chemical resolution methodsare needed. If successful, such methods would suffer from a practicaldisadvantage of wasting half of the prepared material.

As demonstrated in the above examples, the art lacks an efficient methodto generate β-nucleosides. In particular, with sugar precursors carryinga protected hydroxymethyl group at C-4', low selectivity is encounteredduring synthesis of β-isomers or racemization problems occur.Specifically, the art lacks a method of producing stereoselectivelydioxolanes from sugar intermediates carrying a C-2 protectedhydroxymethyl moiety without racemization. Therefore, a generalstereoselective synthesis of biologically active β-nucleoside analoguesis an important goal.

International patent application publication no. WO92/20669 discloses amethod of producing dioxolanes stereoselectively by coupling sugarintermediates carrying C-2 ester moieties with silylated nucleobases andsubsequently reducing the C-2 ester group to the desired hydroxymethylgroup. However, over reduction problems in the pyrimidine base have beendisclosed (Tse et al. Tetrahedron Lett. 1995, 36, 7807-7810).

Nucleoside analogues containing 1,3-dioxolanyl sugars as mimetics of2',3'-dideoxyfuranosyl rings have been prepared by glycosylatingsilylated purine and pyrimidine bases with 1,3-dioxolanes containing aC-2 hydroxymethyl and C-4 acetoxy substituents. The crucial couplingreaction is mediated by trimethylsilytriflate (TMSOT^(f)) oriodotrimethylsilane (TMSI) and produces a mixture of β and α-anomers in1:1 ratio (Kim et al. J. Med. Chem. 1992, 35, 1987-1995 and J. Med.Chem. 1993, 36, 30-37; Belleau et al. Tetrahedron Lett. 1992, 33,6948-6952; and Evans et al. Tetrahedron Asymmetry 1992, 4, 2319-2322).By using metal salts as catalysts the β-nucleoside is favoured (Choi etal. J. Am. Chem. Soc. 1991, 113, 9377-9379) but racemization or loss ofselectivity become a serious limitation (Jin et al. TetrahedronAsymmetry 1993, 4, 2111-2114).

SUMMARY OF THE INVENTION

According to an aspect of the present invention, there is provided aprocess for producing a β-nucleoside analogue compound of formula (III):##STR2## and salts thereof, wherein R₁ is a hydroxyl protecting group;and R₂ is a purine or pyrimidine base or an analogue thereof, theprocess comprising glycosylating said purine or pyrimidine base at atemperature below about -10° C., with an intermediate of formula (II):##STR3## wherein L is halogen.

Subsequent to glycosylation, the compound of formula (III) may thenundergo deprotection of the hydroxyl protecting group R₁ to give a1,3-dioxolane nucleoside analogue of formula (I) ##STR4## wherein R₂ isas previously defined.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a novel method for producing dioxolanenucleoside analogues by coupling sugar precursors carrying a C-2protected hydroxymethyl group with purine or pyrimidine nucleobases inhigh yield and selectivity in favour of the desired β-isomers.

A <<nucleoside>> is defined as any compound which consists of a purineor pyrimidine base or analogue or derivative thereof, linked to apentose sugar.

A <<nucleoside analogue or derivative>> as used hereinafter is acompound containing a 1,3-dioxolane linked to a purine or pyrimidinebase or analog thereof which may be modified in any of the following orcombinations of the following ways: base modifications, such as additionof a substituent (e.g. 5-fluorocytosine) or replacement of one group byan isosteric group (e.g. 7-deazaadenine); sugar modifications, such assubstitution of hydroxyl groups by any substituent or alteration of thesite of attachment of the sugar to the base (e.g. pyrimidine basesusually attached to the sugar at the N-1 site may be, for example,attached at the N-3 or C-6 site and purines usually attached at the N-9site may be, for example, attached at N-7.

A purine or pyrimidine base means a purine or pyrimidine base found innaturally occurring nucleosides. An analogue thereof is a base whichmimics such naturally occurring bases in that its structure (the kindsof atoms and their arrangement) is similar to the naturally occurringbases but may either possess additional or lack certain of thefunctional properties of the naturally occurring bases. Such analoguesinclude those derived by replacement of a CH moiety by a nitrogen atom,(e.g. 5-azapyrimidines, such as 5-azacytosine) or conversely (e.g.,7-deazapurines, such as 7-deazaadenine or 7-deazaguanine) or both (e.g.,7-deaza, 8-azapurines). By derivatives of such bases or analogues aremeant those bases wherein ring substituent are either incorporated,removed, or modified by conventional substituents known in the art, e.g.halogen, hydroxyl, amino, C₁₋₆ alkyl. Such purine or pyrimidine bases,analogs and derivatives are well known to those of skill in the art.

R₁ is a hydroxyl protecting group. Suitable protecting groups includethose described in detail in Protective Groups in Organic Synthesis,Green, John, J. Wiley and Sons, New York (1981). Preferred hydroxylprotecting groups include ester forming groups such as C₁₋₆ acyl i.e.formyl, acetyl, substituted acetyl, propionyl, butanoyl, pivalamido,2-chloroacetyl; aryl substituted C₁₋₆ acyl i.e. benzoyl, substitutedbenzoyl; C₁₋₆ alkoxycarbonyl i.e. methoxycarbonyl; aryloxycarbonyl i.e.phenoxycarbonyl. Other preferred hydroxyl protecting groups includeether forming groups such as C₁₋₆ alkyl i.e. methyl, t-butyl; aryl C₁₋₆alkyl i.e. benzyl, diphenylmethyl any of which is optionally substitutedi.e. with halogen. Particularly preferred hydroxyl protecting groups aret-butoxycarbonyl, benzoyl and benzyl each optionally substituted withhalogen. In a more particularly preferred embodiment the R₁ hydroxylprotecting group is benzyl.

In a preferred embodiment, R₂ is selected from the group consisting of##STR5## wherein R₃ is selected from the group consisting of hydrogen,C₁₋₆ alkyl and C₁₋₆ acyl groups;

R₄ and R₅ are independently selected from the group consisting ofhydrogen, C₁₋₆ alkyl, bromine, chlorine, fluorine, and iodine;

R₆ is selected from the group of hydrogen, halogen, cyano, carboxy, C₁₋₆alkyl, C₁₋₆ alkoxycarbonyl, C₁₋₆ acyl, C₁₋₆ acyloxy, carbamoyl, andthiocarbamoyl; and

X and Y are independently selected from the group of hydrogen, bromine,chlorine, fluorine, iodine, amino, and hydroxyl groups.

In a particularly preferred embodiment R₂ is ##STR6## wherein R₃ and R₄are as previously defined.

In a particularly preferred embodiment R₂ is cytosine or an analogue orderivative thereof. Most preferably R₂ is cytosine, N-acetylcytosine orN-acetyl-5-fluorocytosine.

In preferred embodiments R₃ is H. In another preferred embodiment R₃ isC₁₋₄ acyl such as acetyl.

In preferred embodiments R₄ and R₅ are independently selected fromhydrogen, C₁₋₄ alkyl such as methyl or ethyl and halogen such as F, Cl,I or Br. In particularly preferred embodiments R₄ and R₅ are hydrogen.In another particularly preferred embodiment R₄ and R₅ are F.

In preferred embodiments R₆ is selected from hydrogen, halogen, carboxyand C₁₋₄ alkyl. In particularly preferred embodiments R₆ is H, F or Cland most preferably H.

In preferred embodiments X and Y are independently selected from thegroup of H, F or Cl. In a particularly preferred embodiment X and Y arehydrogen.

L is selected from the group consisting of fluoro, bromo, chloro andiodo.

In a particularly preferred embodiment L is an iodo group. In thisinstance, leaving group (L) may be prepared by displacement of anotherleaving group (L') i.e. acetoxy with Lewis acids containing an iodomoiety. Preferably such Lewis acids have the formula (IV): ##STR7##wherein R₃, R₄ and R₅ are independently selected from the groupconsisting of hydrogen; C₁₋₂₀ alkyl (e.g. methyl, ethyl, ethyl,t-butyl), optionally substituted by halogens (F, Cl, Br, I), C₆₋₂₀alkoxy (e.g., methoxy) or C₆₋₂₀ aryloxy (e.g., phenoxy); C₇₋₂₀ aralkyl(e.g., benzyl), optionally substituted by halogen, C₁₋₂₀ alkyl or C₁₋₂₀alkoxy (e.g., p-methoxybenzyl); C₆₋₂₀ aryl (e.g., phenyl), optionallysubstituted by halogens, C₁₋₂₀ alkyl or C₁₋₂₀ alkoxy; trialkylsilyl;fluoro; bromo; chloro and iodo; and R₆ is selected from the groupconsisting of halogen (F, Cl, Br, I) preferably I (iodo);

L' is a leaving group capable of being displaced by an iodo leavinggroup using a Lewis acid of formula (IV). Suitable leaving groups L'include acyloxy; alkoxy; alkoxycarbonyl; amido; azido; isocyanato;substituted or unsubstituted, saturated or unsaturated thiolates;substituted or unsubstituted, saturated or unsaturated seleno, seleninylor selenonyl compounds; --OR wherein R is a substituted orunsubstituted, saturated or unsaturated alkyl group; a substituted orunsubstituted, aliphatic or aromatic acyl group; a substituted orunsubstituted, saturated or unsaturated alkoxy or aryloxy carbonylgroup, substituted or unsubstituted sulphonyl imidazolide; substitutedor unsubstituted, aliphatic or aromatic amino carbonyl group;substituted or unsubstituted alkyl imidiate group; substituted orunsubstituted, saturated or unsaturated phosphonate; and substituted orunsubstituted, aliphatic or aromatic sulphinyl or sulphonyl group. In apreferred embodiment L' is acetoxy.

In a preferred embodiment, the present invention provides astereoselective process for producing β-nucleoside analogues of formula(III), and salt or ester thereof, by glycosylation of the purine orpyrimidine base or analogue or derivative thereof, with an intermediateof formula (II) as defined previously under low temperature conditions.Preferably, the glycosylation reaction takes place at temperatures below-10° C. i.e. about -10 to -100° C. and more preferably below -20° C. Ina most preferred embodiment the glycosylation reaction occurs betweenabout -20 to -78° C.

The intermediate of formula II is reacted with a silylated purine orpyrimidine base, conveniently in a suitable organic solvent such as ahydrocarbon, for example, toluene, a halogenated hydrocarbon such asdichloromethane (DCM), a nitrile, such as acetonitrile, an amide such asdimethylformamide, an ester, such as ethyl acetate, an ether such astetrahydrofuran, or a mixture thereof, at low temperatures, such as -40°C. to -78° C. Silylated purine or pyrimidine bases or analogues andderivatives thereof may be prepared as described in WO92/20669, theteaching of which is incorporated herein by reference. Such silylatingagents are 1,1,1,3,3,3-hexamethyldisilazane, trimethylsilyl triflate,t-butyldimethylsilyl triflate or trimethylsilyl chloride, with acid orbase catalyst, as appropriate. The preferred silylating agent is1,1,1,3,3,3,-hexamethyldisilazane.

To form the compound of formula (I), appropriate deprotecting conditionsinclude methanolic or ethanolic ammonia or a base such as potassiumcarbonate in an appropriate solvent such as methanol or tetrahydrofuranfor N-4 deacetytion.

Transfer deacetylation hydrogenolysis with a hydrogen donor such ascyclohexene or ammonium formate in the presence of a catalyst such aspalladium oxide over charcoal are appropriate for the removal of the5'-aryl group.

It will be appreciated that the intermediate of formula (II) isconstituted by intermediates IIa and IIb: ##STR8##

It will be further appreciated that, if the glycosylation step iscarried out using equimolar amounts of intermediates IIa and IIb, aracemic mixture of β-nucleosides of formula I is obtained.

It will be apparent to those of skill in the art that separation of theresulting diastereomic mixture, for example after the coupling reactionbetween compounds of formula II and a silylated base, can be achieved bychromatography on silica gel or crystallization in an appropriatesolvent (see, for example: J. Jacques et al. Enantiomers, Racemates andResolutions, pp 251-369, John Wiley and Sons, New York 1981).

However, it is preferred that glycosylation is effected using anoptically pure compound of either formula IIa or IIb, thereby producingthe desired nucleoside analog in high optical purity.

The compounds of formula IIa or IIb exist as mixture of twodiastereomers epimeric at the C-4 centre. We have now found that asingle diastereomer, as well as any mixture of the diastereomerscomprising the compounds of formula IIa, react with silylated bases toproduce β-L nucleosides in high optical purity. The base at C-4 havingthe cis-stereochemistry relative to the hydroxymethyl moiety at C-2. Therate of the reaction of the two diastereomers of formula IIa withsilylated bases may however, be different. Similar findings exist forthe intermediates of formula IIb for the synthesis of β-D nucleosides.

In a preferred embodiment, the present invention provides a step forproducing anomeric iodides of formula II by reacting known anomeric2S-benzyloxymethyl-1,3-dioxolane-4S and -4R acetoxy derivatives offormula (V) with iodotrimethylsilane or diiodosilane at low temperatures(-78° C.) prior to glycosylation with silylated pyrimidine or purinebase or analogue or derivative thereof (Scheme 1). ##STR9##

Reagents and conditions:

i) ##STR10## ii) MeOH/LiOH; iii) Column separation;

iv) Pb(OAc)₄ /MeCN/Py/2 h/RT/80%; and

v) TMSI or SiH₂ I₂ /CH₂ Cl₂ /-78° C.

Suitable methods for producing the anomeric acetoxy intermediate (VI)will be readily apparent to those skilled in the art and includeoxidative degradation of benzyloxymethylacetals derived from L-ascorbicacid (Belleau et al. Tetrahedron Lett. 1992, 33, 6949-6952) orD-mannitol (Evans et al. Tetrahedron Asymmetry 1993, 4, 2319-2322).

We have also found that the known2S-benzyloxymethyl-1,3-dioxolane-4S-carboxyclic acid (V) can begenerated in preference to its 2S,4R isomer by reacting commerciallyavailable 2,2-dimethyl-1,3-dioxolane-4S-carboxylic acid with a protectedderivative of hydroxyacetaldehyde, such as benzyloxyacetaldehyde, underacidic conditions.

In the diastereoselective process of this invention, there is alsoprovided the following intermediates:

2S-Benzyloxymethyl-4R-iodo-1,3 dioxolane and2S-Benzyloxymethyl-4S-iodo-1,3 dioxolane;

β-L-5'-Benzyl-2'-deoxy-3'-oxa-N-4-acetyl-cytidine;

β-L-5'-Benzyloxy-2'-deoxy-3'-oxacytidine;

β-L-5'-Benzyl-2'-deoxy-3'-oxa-5-fluoro-N4-acetyl-cytidine; and

β-L-5'-Benzyl-2'-deoxy-3'-oxa-5-fluorocytidine.

EXAMPLE 1A 2S-Benzyloxymethyl-4R-iodo-1,3 dioxolane and2S-Benzyloxymethyl-4S-iodo-1,3 dioxolane (compound #1) ##STR11##

A mixture consisting of 2S-benzyloxymethyl-4S acetoxy-1,3 dioxolane and2S-benzyloxymethyl-4R-acetoxy-1,3 dioxolane in 1:2 ratio (6 g; 23.8mmol) was dried by azeotropic distillation with toluene in vacuo. Afterremoval of toluene, the residual oil was dissolved in drydichloromethane (60 ml) and iodotrimethylsilane (3.55 ml; 1.05 eq) wasadded at -78° C., under vigorous stirring. The dry-ice/acetone bath wasremoved after addition and the mixture was allowed to warm up to roomtemperature (15 min.). The ¹ H NMR indicated the formation of2S-benzyloxymethyl-4R-iodo-1,3-dioxolane and2S-benzyloxymethyl-4S-iodo-1,3 dioxolane.

¹ H NMR (300 MHz, CDCl₃) δ 3.65-4.25 (2H,m); 4.50-4.75 (4H,m) 5.40-5.55(1H, overlapping triplets); 6.60-6.85 (1H, d of d); 7.20-7.32 (5H,m).

EXAMPLE 1b 2S-Benzyloxymethyl-4R-iodo-1,3 dioxolane and2S-Benzyloxymethyl-4S-iodo-1,3 dioxolane (compound #1) ##STR12##

A mixture consisting of 2S-benzyloxymethyl-4S acetoxy-1,3 dioxolane and2S-benzyloxymethyl-4R-acetoxy-1,3 dioxolane in 1:2 ratio (6 g; 23.8mmol) was dried by azeotropic distillation with toluene in vacuo. Afterremoval of toluene, the residual oil was dissolved in drydichloromethane (60 ml) and diiodosilane (2.4 ml; 1.05 eq) was added at-78° C., under vigorous stirring. The dry-ice/acetone bath was removedafter addition and the mixture was allowed to warm up to roomtemperature (15 min.). The ¹ H NMR indicated the formation of2S-benzyloxymethyl-4R-iodo-1,3-dioxolane and2S-benzyloxymethyl-4S-iodo-1,3 dioxolane.

¹ H NMR (300 MHz, CDCl₃) δ 3.65-4.25 (2H,m); 4.50-4.75 (4H,m) 5.40-5.55(1H, overlapping triplets); 6.60-6.85 (1H, d of d); 7.20-7.32 (5H,m).

EXAMPLE 2 β-L-5'-Benzyl-2'-deoxy-3'-oxa-N-4-acetyl-cytidine (compound#2) ##STR13##

The previously prepared iodo intermediate (example 1) indichloromethane, was cooled down to -78° C. Persylilated N-acetylcytosine (1.1 eq) formed by reflux in 1,1,1,3,3,3-hexamethyl disilazane(HMDS) and ammonium sulphate followed by evaporation of HMDS wasdissolved in 30 ml of dichloromethane and was added to the iodointermediate. The reaction mixture was maintained at -78° C. for 1.5hours then poured onto aqueous sodium bicarbonate and extracted withdichloromethane (2×25 ml). The organic phase was dried over sodiumsulphate, the solid was removed by filtration and the solvent wasevaporated in vacuo to produce 8.1 g of a crude mixture. Based on ¹ HNMR analysis, the β-L-5'-benzyl-2'-deoxy-3'-oxacytidine and its α-Lisomer were formed in a ratio of 5:1 respectively. This crude mixturewas separated by chromatography on silica-gel (5% MeOH in EtOAc) togenerate the pure β-L (cis) isomer (4.48 g). Alternatively,recrystallization of the mixture from ethanol produces 4.92 g of pure βisomer and 3.18 g of a mixture of β and α-isomers in a ratio of 1:1.

¹ H NMR (300 MHz, CDCl₃) δ 2.20 (3H,S,Ac); 3.87 (2H,m,H-5'), 4.25(2H,m,H-2'); 4.65 (2H,dd,OCH₂ Ph); 5.18 (1H,t,H-4'); 6.23 (1H,m,H-1');7.12 (1H,d,H-5); 7.30-7.50 (5H,m,Ph); 8.45 (2H,m,NH+H-6).

EXAMPLE 3 β-L-5'-Benzyloxy-2'-deoxy-3'-oxacytidine (compound #3)##STR14##

The protected β-L isomer (4.4 g) of example 2 was suspended in saturatedmethanolic ammonia (250 ml) and stirred at room temperature for 18 hoursin a closed vessel. The solvents were then removed in vacuo to affordthe deacetylated nucleoside in pure form.

¹ H NMR (300 MHz, CDCl₃) δ 3.85 (2H,m,H-5'); 4.20 (2H,m,H-2'); 4.65(2H,dd,OCH₂ Ph); 5.18 (1H,t,H-4'); 5.43 (1H,d,H-5); 5.50-5.90(2H,br.S,NH₂); 6.28 (1H,m,H-1'); 7.35-7.45 (5H,m,Ph); 7.95 (1H,d,H-6).

EXAMPLE 4 β-L-2'-deoxy-3'-oxacytidine (compound #4) ##STR15##

β-L-5'-Benzyl-2'-deoxy-3'-oxacytidine from the previous example, wasdissolved in EtOH (200 ml) followed by addition of cyclohexene (6 ml)and palladium oxide (0.8 g). The reaction mixture was refluxed for 7hours then it was cooled and filtered to remove solids. The solventswere removed from the filtrate by vacuum distillation. The crude productwas purified by flash chromatography on silica-gel (5% MeOH in EtOAc) toyield a white solid (2.33 g; 86% overall yield, α_(D) ²² =-46.7°(c=0.285; MeOH) m.p.=192-194° C.

¹ H NMR (300 MHz,DMSO-d₆) δ 3.63 (2H,dd,H-5'); 4.06 (2H,m,H-2'); 4.92(1H,t,H-4'); 5.14 (1H,t,OH); 5.70 (1H,d,H-5); 6.16 (2H,dd,H-1');7.11-7.20 (2H,brS,NH₂); 7.80 (1H,d,H-6) ¹³ C NMR (75 MHz,DMSO-d₆) δ 59.5(C-2'); 70.72 (C-5'); 81.34 (C-4'); 93.49 (C-1'); 104.49 (C-5); 140.35(C-4); 156.12 (C-6); 165.43 (C-2).

EXAMPLE 5 β-L-5'-Benzyl-2'-deoxy-3'-oxa-5-fluoro-N4-acetyl-cytidine(compound #5) ##STR16##

The previously prepared iodo derivatives (example 1) in dichloromethane,was cooled down to -78° C. Persylilated N-acetyl-5-fluorocytosine (1.05eq) formed by reflux in 1,1,1,3,3,3-hexamethyldisilazane (HMDS) andammonium sulphate followed by evaporation of HMDS was dissolved in 20 mlof dichloromethane (DCM) and was added to the iodo intermediate. Thereaction mixture was maintained at -78° C. for 1.5 hours then pouredonto aqueous sodium bicarbonate and extracted with dichloromethane (2×25ml). The organic phase was dried over sodium sulphate, the solid wasremoved by filtration and the solvent was evaporated in vacuo to produce8.1 g of a crude mixture. Based on ¹ H NMR analysis, theβ-L-5'-benzyl-2'-deoxy-3'-oxa-5-fluoro-N4-acetyl-cytidine and its α-Lisomer were formed in a ratio of 5:1 respectively. This crude mixturewas separated by chromatography on silica-gel (5% MeOH in EtOAc) togenerate the pure β-L (cis) isomer (4.48 g). Alternatively,recrystallization of the mixture from ethanol produces 4.92 g of pure βisomer and 3.18 g of a mixture of β and α-isomers in a ratio of 1:1.

¹ H NMR (300 MHz, CDCl₃) δ 2.20 (3H,S,Ac); 3.87 (2H,m,H-5'), 4.25(2H,m,H-2'); 4.65 (2H,dd,OCH₂ Ph); 5.18 (1H,t,H-4'); 6.23 (1H,m,H-1');7.12 (1H,d,H-5); 7.30-7.50 (5H,m,Ph⁻); 8.45 (2H,m,NH+H-6).

EXAMPLE 6 β-L-5'-Benzyl-2'-deoxy-3'-oxa-5-fluorocytidine (compound #6):##STR17##

The crude mixture from previous step (example 5) was suspended inmethanolic ammonia (100 ml) and stirred for 18 hours at room temperaturein a closed reaction vessel. The solvents were removed in vacuo toafford the deacetylated mixture which was separated by flashchromatography on silica gel (2% to 3% MeOH in EtOAc) to yield 1.21 gpure β isomer (yield 45% with respect to this isomer).

EXAMPLE 7 β-L-2'-deoxy-3'-oxa-5-fluorocytidine (compound #7) ##STR18##

The deacetylated pure β-L isomer (900 mg; 2.8 mmol) prepared asdescribed in example 6 was dissolved in EtOH (40 ml) followed byaddition of cyclohexene (3 ml) and palladium oxide catalyst (180 mg).The reaction was refluxed for 24 hours and the catalyst was removed byfiltration. The solvents were removed from the filtrate by vacuumdistillation. The crude product was purified by flash chromatography onsilica-gel (5% to 7% MeOH in EtOAc) to yield a white solid (530 mg ; 82%yield). (α²² _(D))=-44.18° (c=0.98; MeOH).

¹ H NMR (300 MHz, DMSO-d₆); δ 3.62-3.71 (2H,m,H-5'); 4.03-4.13(2H;m,H-2'); 4.91 (1H,t,H-4'); 5.32 (1H,t,OH); 6.11 (1H;t;H-1');7.53-7.79 (2H,b,NH₂); 8.16 (1H;d,H-6); ¹³ C NMR (75 MHz, DMSO-d₆); δ59.34 (C-2'); 70.68 (C-5'); 80.78 (C-4'); 104.53-(C-1'); 124.90, 125.22(C-4); 134.33, 136.73 (C-5); 153.04 (C-2); 156.96, 157.09 (C-6).

EXAMPLE 8 Isomeric purity determination of β-L-2'-deoxy-3'-oxacytidinenucleoside analogues:

The determination of the isomeric purity (β-L versus α-L and β-L versusβ-D isomers) was determined on a Waters HPLC system consisting of a 600controller pump for solvent delivery, 486 uv detector, 412 WISP autosampler and a 740 Waters integrator module. An analytical chiral reversephase cyclobond I RSP column (Astec, 4.6×250 mm i.d.) was used andpacked by the manufacturer with β-cyclodextrin derivatized withR'S-hydroxypropyl ether. The mobile phase consisted of acetonitrile (A)and water containing 0.05% triethylamine (B) with the pH adjusted to7.05 by glacial acetic acid. The column was operated under isocraticconditions at 0° C. using a mixture of 5% A and 95% B. Such conditionsare modifications of those reported in DiMarco et al. (J.Chromatography, 1993, 645, 107-114). The flow rate was 0.22 ml/min andthe pressure was maintained at 648-660 psi. Detection of nucleosides wasmonitored by uv absorption at 215 and 265 nm. Samples of β-D isomer andracemic compounds were prepared as reported (Belleau et al. TetrahedronLett 1992, 33, 6948-6952) and used for internal references andco-injection.

Under these conditions the isomeric purity of compound #4 producedaccording to example 4 was>99% and that of compound #7 according toexample 7, was>96%.

The isomeric purity of dioxolane nucleosides having been preparedaccording to the general scheme 2, under varying conditions i.e.temperature and Lewis acid is represented in table 1 below. Thoseprepared at temperatures above-10° C. exhibited reducedstereoselectivity.

                  TABLE 1    ______________________________________    Scheme 2    1 #STR19##    2 #STR20##    3 #STR21##    Base        Lewis acid                          Temperature (°C.)                                       Cis:trans    ______________________________________    5F-N(Ac)-cytosine                TMSI      a:-78 b:-78  8:1    5F-N(Ac)-cytosine                SiH.sub.2 I.sub.2                          a:-78 b:-78  7:2    N(Ac)-cytosine                TMSI      a:-78 b:-78  5:1    ______________________________________     note: all reactions in DCM solvent and bases silylated with HMDS.

We claim:
 1. A process for producing a β-nucleoside analogue compound offormula (IIIa) or (IIIb): ##STR22## and salts thereof, wherein R₁ is ahydroxyl protecting group and R₂ is a purinyl or pyrimidinyl group or aderivative thereof, the process comprisingglycosylating a purine orpyrimidine base or derivative thereof at a temperature of about -10° C.or less with a compound of formula (II); ##STR23## wherein L is halogen;to produce said β-nucleoside analogue.
 2. The process according to claim1, wherein L is iodo.
 3. The process according to claim 2, wherein R₁ isbenzyl.
 4. The process according to claim 1, wherein R₂ is selected fromthe group consisting of ##STR24## wherein R₃ is selected from the groupconsisting of hydrogen, C₁₋₆ alkyl and a group of formula RC(O) whereinR is hydrogen or C₁₋₅ alkyl;R₄ and R₅ are each independently selectedfrom the group consisting of hydrogen, C₁₋₆ alkyl, bromine, chlorine,fluorine and iodine; R₆ is selected from the group consisting ofhydrogen, halogen, cyano, carboxy, C₁₋₆ alkyl, C₁₋₆ alkoxycarbonyl, agroup of formula RC(O) wherein R is hydrogen or C₁₋₅ alkyl, a group offormula RC(O)O wherein R is hydrogen or C₁₋₅ alkyl, carbamoyl andthiocarbamoyl; and X and Y are each independently selected from thegroup consisting of hydrogen, bromine, chlorine, fluorine, iodine, aminoand hydroxyl.
 5. The process according to claim 1, wherein R₂ is##STR25## wherein R₃ is selected from the group consisting of hydrogen,C₁₋₆ alkyl and a group of formula RC(O) wherein R is hydrogen or C₁₋₅alkyl; andR₄ is selected from the group consisting of hydrogen, C₁₋₆alkyl, bromine, chlorine, fluorine and iodine.
 6. The process accordingto claim 5, wherein R₃ is hydrogen or acetyl and R₄ is hydrogen orfluorine.
 7. The process according to claim 1, wherein saidglycosylating step is conducted at a temperature of about -15° C. orless.
 8. The process according to claim 7, wherein L is iodo.
 9. Theprocess according to claim 8, wherein R₁ is benzyl.
 10. The processaccording to claim 1, wherein said glycosylating step is conducted at atemperature of about -20° C. or less.
 11. The process according to claim10, wherein L is iodo.
 12. The process according to claim 11, wherein R₁is benzyl.
 13. The process according to claim 1, wherein saidglycosylating step is conducted at a temperature of about -50° C. orless.
 14. The process according to claim 13, wherein L is iodo.
 15. Theprocess according to claim 14, wherein R₁ is benzyl.
 16. The processaccording to claim 1, wherein said glycosylating step is conducted at atemperature of about -78° C.
 17. The process according to claim 16,wherein L is iodo.
 18. The process according to claim 17, wherein R₁ isbenzyl.
 19. The process according to claim 1, further comprisingremoving the hydroxyl protecting group.
 20. The process according toclaim 1, wherein the compound of formula (II) is prepared by reacting acompound of formula (II') ##STR26## wherein L' is a leaving group; witha Lewis acid of formula (IV) ##STR27## wherein R₃, R₄ and R₅ are eachindependently selected from the group consisting of hydrogen;C₁₋₂₀ alkylwhich is unsubstituted or substituted by halogen, C₁₋₂₀ alkoxy or C₆₋₂₀aryloxy; C₇₋₂₀ aralkyl which is unsubstituted or substituted by halogen,C₁₋₂₀ alkyl or C₁₋₂₀ alkoxy; C₆₋₂₀ aryl which is unsubstituted orsubstituted by halogen, C₁₋₂₀ alkyl or C₁₋₂₀ alkoxy; trialkylsilyl,fluoro, bromo, chloro and iodo; and R₆ is halogen.
 21. The processaccording to claim 20, wherein the Lewis acid is TMSI or SiH₂ I₂.. 22.The process according to claim 20, wherein the Lewis acid is TMSI.